Tobacco having altered leaf properties and methods of making and using

ABSTRACT

This disclosure provides tobacco plants having a mutation in PR50 and transgenic tobacco plants containing a PR50 RNAi, and methods of making and using such plants.

CLAIM OF PRIORITY

This application claims priority to U.S. Provisional Application Ser.No. 62/019,936, filed on Jul. 2, 2015, the entire contents of which arehereby incorporated by reference.

TECHNICAL FIELD

This disclosure generally relates to transgenic or mutant Nicotianatabacum plants and methods of making and using such plants.

BACKGROUND

Nicotine is an abundant alkaloid (90-95%) present in cultivated tobacco.The remaining alkaloid fraction is primarily comprised of threeadditional alkaloids: nornicotine, anabasine, and anatabine. Thisdisclosure describes methods of modulating the expression and/oractivity of PR50 to thereby reduce the amount of nicotine and otheralkaloids in the leaf.

SUMMARY

Provided herein are transgenic tobacco plants containing a PR50 RNAi andtobacco plants having a mutation in the gene encoding PR50, as well asmethods of making and using such plants.

In one aspect, a RNA nucleic acid molecule is provided that includes afirst nucleic acid between 15 and 500 nucleotides in length and a secondnucleic acid between 15 and 500 nucleotides in length. Generally, thefirst nucleic acid has a region of complementarity to the second nucleicacid, and the first nucleic acid comprises at least 15 contiguousnucleotides of the sequence shown in SEQ ID NO:1.

In some embodiments, the second nucleic acid hybridizes under stringentconditions to a portion of the sequence shown in SEQ ID NO:1. In someembodiments, the region of complementarity is at least 19 nucleotides inlength. In some embodiments, the region of complementarity is at least100 nucleotides in length. In some embodiments, a nucleic acid moleculeas described herein can further include a spacer nucleic acid betweenthe first nucleic acid and the second nucleic acid.

In another aspect, a construct is provided that includes a first RNAnucleic acid molecule having a length of 15 to 500 nucleotides andhaving at least 95% sequence identity to a nucleic acid shown in SEQ IDNO:1. In some embodiments, the construct can further include a secondRNA nucleic acid molecule that has complementarity to the first RNAnucleic acid molecule. In some embodiments, the construct can furtherinclude a spacer nucleic acid between the first and second RNA nucleicacid molecule.

In still another aspect, a method of making a Nicotiana tabacum plant isprovided. Such a method typically includes transforming N. tabacum cellswith a nucleic acid molecule as described herein or a construct asdescribed herein to produce transgenic N. tabacum cells; regeneratingtransgenic N. tabacum plants from the transgenic N. tabacum cells; andselecting at least one transgenic N. tabacum plant that comprises thenucleic acid molecule or the construct.

In some embodiments, such a method further includes identifying at leastone transgenic N. tabacum plant having reduced amount of nicotinerelative to a N. tabacum plant not transformed with the nucleic acidmolecule. In some embodiments, such a method further includesidentifying at least one transgenic N. tabacum plant that, when materialfrom the at least one transgenic N. tabacum plant is cured, exhibits areduced amount of at least one TSNA relative to cured material from a N.tabacum plant not transformed with the nucleic acid molecule. In someembodiments, leaf from the selected transgenic N. tabacum plant exhibitscomparable or better quality than leaf from the non-transformed N.tabacum plant. In some embodiments, the N. tabacum plant is a Burleytype, a dark type, a flue-cured type, or an Oriental type.

In another aspect, a transgenic Nicotiana tabacum plant is provided thatincludes a vector. Generally, the vector includes a RNA nucleic acidmolecule having a length of 15 to 500 nucleotides and having at least95% sequence identity to a PR50 nucleic acid shown in SEQ ID NO:1. Insome embodiments, the plant exhibits reduced amount of nicotine in theleaf relative to leaf from a N. tabacum plant lacking the nucleic acidmolecule. In some embodiments, when material from the at least onetransgenic N. tabacum plant is cured, it exhibits a reduced amount of atleast one TSNA relative to cured material from a N. tabacum plantlacking the nucleic acid molecule. In some embodiments, leaf from theplant exhibits comparable or better quality than leaf from a N. tabacumplant lacking the nucleic acid molecule.

In still another aspect, cured leaf is provided from any of thetransgenic N. tabacum plants described herein. In yet another aspect, atobacco product is provided that includes cured leaf as describedherein. Representative tobacco products include, without limitation,cigarettes, smokeless tobacco products, tobacco-derived nicotineproducts (e.g., tobacco-derived nicotine pieces for use in the mouth),cigarillos, non-ventilated recess filter cigarettes, vented recessfilter cigarettes, cigars, electronic cigarettes, electronic cigars,electronic cigarillos, e-vapor devices, snuff, pipe tobacco, cigartobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shreddedtobacco, and cut tobacco.

In one aspect, a method of making a Nicotiana tabacum plant is provided.Such a method typically includes inducing mutagenesis in N. tabacumcells to produce mutagenized N. tabacum cells; obtaining one or more N.tabacum plants from the mutagenized N. tabacum cells; and identifying atleast one of the N. tabacum plants that comprises a mutated PR50sequence.

In some embodiments, such a method can further include identifying atleast one of the N. tabacum plants that exhibits reduced amounts ofnicotine relative to a N. tabacum plant lacking a mutated PR50. In someembodiments, such a method can further include identifying at least oneof the N. tabacum plants that, when material from the at least one plantis cured, exhibits a reduced amount of at least one TSNA relative tocured material from a N. tabacum plant lacking a mutated PR50. In someembodiments, leaf from the mutant N. tabacum plant exhibits comparableor better quality than leaf from the plant lacking a mutated PR50sequence. In some embodiments, the N. tabacum plant is a Burley type, adark type, a flue-cured type, or an Oriental type.

In another aspect, a variety of Nicotiana tabacum is provided.Generally, the variety includes plants having a mutation in anendogenous nucleic acid, where the wild type endogenous nucleic acidencodes the PR50 sequence shown in SEQ ID NO:2. In some embodiments,leaf from the mutant plants exhibits a reduced amount of nicotinerelative to leaf from a plant lacking the mutation. In some embodiments,material from the mutant plants, when cured, exhibits a reduced amountof at least one TSNA relative to cured material from a plant lacking themutation. In some embodiments, leaf from the mutant N. tabacum plantexhibits comparable or better quality than leaf from the plant lacking amutated PR50 sequence.

In still another aspect, cured leaf is provided from any of the N.tabacum varieties described herein. In yet another aspect, a tobaccoproduct is provided that includes cured leaf described herein.Representative tobacco products include, without limitation, cigarettes,smokeless tobacco products, tobacco-derived nicotine products (e.g.,tobacco-derived nicotine pieces for use in the mouth), cigarillos,non-ventilated recess filter cigarettes, vented recess filtercigarettes, cigars, electronic cigarettes, electronic cigars, electroniccigarillos, e-vapor devices, snuff, pipe tobacco, cigar tobacco,cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, andcut tobacco.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1 is an alignment of the PR50 nucleotide sequence and its homologs(SEQ ID NOs: 2 and 11-14 (top to bottom)).

FIG. 2 is an alignment of the PR50 amino acid sequence and its homologs(SEQ ID NOs: 3 and 15-18 (top to bottom)).

FIG. 3 is a graph showing the nicotine levels in T848 transgenic orcontrol plants (T1 and T2 generations) following growth in the field.

FIG. 4 is a graph showing nicotine levels in TN90 PMT RNAi, TN90 PR50RNAi, LA Burley 21, and TN90 wild type varieties.

DETAILED DESCRIPTION

PR50 is a cDNA that is differentially expressed in roots of Nicotianatabacum cv Burley 21 during the early stages of alkaloid biosynthesis.See, for example, Wang et al., 2000, Plant Sci., 158:19 -32. PR50 hasabout 88-93% sequence identity at the nucleic acid level, and 93-97%sequence identity at the amino acid level, to a 40S ribosomal proteinfrom Solanum spp. The present disclosure describes several differentapproaches that can be used to significantly reduce nicotine levels intobacco leaf while maintaining leaf quality.

PR50 Nucleic Acids and Polypeptides

A nucleic acid encoding PR50 from N. tabacum is shown in SEQ ID NO: 1(genomic) and SEQ ID NO:2 (cDNA). A nucleic acid encoding a PR50homologue from N. tabacum is shown in SEQ ID NO: 23 (cDNA). FIG. 1 is analignment of the PR50 nucleotide sequence and several PR50 homologs.Unless otherwise specified, nucleic acids referred to herein can referto DNA and RNA, and also can refer to nucleic acids that contain one ormore nucleotide analogs or backbone modifications. Nucleic acids can besingle stranded or double stranded, and linear or circular, both ofwhich usually depend upon the intended use.

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is free of sequences that naturally flank one or both endsof the nucleic acid in the genome of the organism from which theisolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNAfragment produced by PCR or restriction endonuclease digestion). Such anisolated nucleic acid molecule is generally introduced into a vector(e.g., a cloning vector, or an expression vector) for convenience ofmanipulation or to generate a fusion nucleic acid molecule, discussed inmore detail below. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant or asynthetic nucleic acid molecule.

The sequence of the PR50 polypeptide from N. tabacum is shown in SEQ IDNO: 3, and the sequence of the PR50 homologue polypeptide from N.tabacum is shown in SEQ ID NO:24. FIG. 2 is an alignment of the PR50amino acid sequence and several PR50 homologs (SEQ ID NOs: 3 and 15-18(top to bottom)). As used herein, a “purified” polypeptide is apolypeptide that has been separated or purified from cellular componentsthat naturally accompany it. Typically, the polypeptide is considered“purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%,95%, or 99%) by dry weight, free from the polypeptides and naturallyoccurring molecules with which it is naturally associated. Since apolypeptide that is chemically synthesized is, by nature, separated fromthe components that naturally accompany it, a synthetic polypeptide is“purified.”

Nucleic acids can be isolated using techniques well known in the art.For example, nucleic acids can be isolated using any method including,without limitation, recombinant nucleic acid technology, and/or thepolymerase chain reaction (PCR). General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleicacid techniques include, for example, restriction enzyme digestion andligation, which can be used to isolate a nucleic acid. Isolated nucleicacids also can be chemically synthesized, either as a single nucleicacid molecule or as a series of oligonucleotides.

Polypeptides can be purified from natural sources (e.g., a biologicalsample) by known methods such as DEAE ion exchange, gel filtration, andhydroxyapatite chromatography. A polypeptide also can be purified, forexample, by expressing a nucleic acid in an expression vector. Inaddition, a purified polypeptide can be obtained by chemical synthesis.The extent of purity of a polypeptide can be measured using anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

Nucleic acids can be detected using any number of amplificationtechniques (see, e.g., PCR Primer: A Laboratory Manual, 1995,Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;and 4,965,188) with an appropriate pair of oligonucleotides (e.g.,primers). A number of modifications to the original PCR have beendeveloped and can be used to detect a nucleic acid. Nucleic acids alsocan be detected using hybridization.

Polypeptides can be detected using antibodies. Techniques for detectingpolypeptides using antibodies include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Anantibody can be polyclonal or monoclonal. An antibody having specificbinding affinity for a polypeptide can be generated using methods wellknown in the art. The antibody can be attached to a solid support suchas a microtiter plate using methods known in the art. In the presence ofa polypeptide, an antibody-polypeptide complex is formed.

Detection (e.g., of an amplification product, a hybridization complex,or a polypeptide) is oftentimes accomplished using detectable labels.The term “label” is intended to encompass the use of direct labels aswell as indirect labels. Detectable labels include enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials.

A construct, sometimes referred to as a vector, containing a nucleicacid (e.g., a coding sequence or a RNAi nucleic acid molecule) isprovided. Constructs, including expression constructs (or expressionvectors), are commercially available or can be produced by recombinantDNA techniques routine in the art. A construct containing a nucleic acidcan have expression elements operably linked to such a nucleic acid, andfurther can include sequences such as those encoding a selectable marker(e.g., an antibiotic resistance gene). A construct can encode a chimericor fusion polypeptide (i.e., a first polypeptide operatively linked to asecond polypeptide). Representative first (or second) polypeptides arethose that can be used in purification of the other (i.e., second (orfirst), respectively) polypeptide including, without limitation, 6× Histag or glutathione S-transferase (GST).

Expression elements include nucleic acid sequences that direct andregulate expression of nucleic acid coding sequences. One example of anexpression element is a promoter sequence. Expression elements also caninclude introns, enhancer sequences, response elements, or inducibleelements that modulate expression of a nucleic acid. Expression elementscan be of bacterial, yeast, insect, mammalian, or viral origin, andvectors can contain a combination of elements from different origins. Asused herein, operably linked means that a promoter or other expressionelement(s) are positioned in a vector relative to a nucleic acid in sucha way as to direct or regulate expression of the nucleic acid (e.g.,in-frame).

Constructs as described herein can be introduced into a host cell. Manymethods for introducing nucleic acids into host cells, both in vivo andin vitro, are well known to those skilled in the art and include,without limitation, electroporation, calcium phosphate precipitation,polyethylene glycol (PEG) transformation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer. As usedherein, “host cell” refers to the particular cell into which the nucleicacid is introduced and also includes the progeny or potential progeny ofsuch a cell. A host cell can be any prokaryotic or eukaryotic cell. Forexample, nucleic acids can be introduced into bacterial cells such as E.coli, or into insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art.

RNA Interfering Nucleic Acids and Constructs Containing Same

RNA interference (RNAi), also called post-transcriptional gene silencing(PTGS), is a biological process in which RNA molecules inhibit geneexpression, typically by causing the destruction of specific mRNAmolecules. Without being bound by theory, it appears that, in thepresence of an antisense RNA molecule that is complementary to anexpressed message (i.e., a mRNA), the two strands anneal to generatelong double-stranded RNA (dsRNA), which is digested into short (<30nucleotide) RNA duplexes, known as small interfering RNAs (siRNAs), byan enzyme known as Dicer. A complex of proteins known as the RNA InducedSilencing Complex (RISC) then unwinds siRNAs, and uses one strand toidentify and thereby anneal to other copies of the original mRNA. RISCcleaves the mRNA within the complementary sequence, leaving the mRNAsusceptible to further degradation by exonucleases, which effectivelysilences expression of the encoding gene.

Several methods have been developed that take advantage of theendogenous machinery to suppress the expression of a specific targetgene and a number of companies offer RNAi design and synthesis services(e.g., Life Technologies, Applied Biosystems). In transgenic plants, theuse of RNAi can involve the introduction of long dsRNA (e.g., greaterthan 50 bps) or siRNAs (e.g., 12 to 23 bps) that have complementarity tothe target gene, both of which are processed by the endogenousmachinery. Alternatively, the use of RNAi can involve the introductionof a small hairpin RNA (shRNA); shRNA is a nucleic acid that includesthe sequence of the two desired siRNA strands, sense and antisense, on asingle strand, connected by a “loop” or “spacer” nucleic acid. When theshRNA is transcribed, the two complementary portions annealintra-molecularly to form a “hairpin,” which is recognized and processedby the endogenous machinery.

A RNAi nucleic acid molecule as described herein is complementary to atleast a portion of a target mRNA (i.e., a PR50 mRNA), and typically isreferred to as an “antisense strand”. Typically, the antisense strandincludes at least 15 contiguous nucleotides of the

DNA sequence (e.g., the PR50 nucleic acid sequence shown in SEQ ID NO:1,2 or 23); it would be appreciated that the antisense strand has the “RNAequivalent” sequence of the DNA (e.g., uracils instead of thymines;ribose sugars instead of deoxyribose sugars).

A RNAi nucleic acid molecule can be, for example, 15 to 500 nucleotidesin length (e.g., 15 to 50, 15 to 45, 15 to 30, 16 to 47, 16 to 38, 16 to29, 17 to 53, 17 to 44, 17 to 38, 18 to 36, 19 to 49, 20 to 60, 20 to40, 25 to 75, 25 to 100, 28 to 85, 30 to 90, 15 to 100, 15 to 300, 15 to450, 16 to 70, 16 to 150, 16 to 275, 17 to 74, 17 to 162, 17 to 305, 18to 60, 18 to 75, 18 to 250, 18 to 400, 20 to 35, 20 to 60, 20 to 80, 20to 175, 20 to 225, 20 to 325, 20 to 400, 20 to 475, 25 to 45, 25 to 65,25 to 100, 25 to 200, 25 to 250, 25 to 300, 25 to 350, 25 to 400, 25 to450, 30 to 280, 35 to 250, 200 to 500, 200 to 400, 250 to 450, 250 to350, or 300 to 400 nucleotides in length).

In some embodiments, the “antisense strand” (e.g., a first nucleic acid)can be accompanied by a “sense strand” (e.g., a second nucleic acid),which is complementary to the antisense strand. In the latter case, eachnucleic acid (e.g., each of the sense and antisense strands) can bebetween 15 and 500 nucleotides in length (e.g., between 15 to 50, 15 to45, 15 to 30, 16 to 47, 16 to 38, 16 to 29, 17 to 53, 17 to 44, 17 to38, 18 to 36, 19 to 49, 20 to 60, 20 to 40, 25 to 75, 25 to 100, 28 to85, 30 to 90, 15 to 100, 15 to 300, 15 to 450, 16 to 70, 16 to 150, 16to 275, 17 to 74, 17 to 162, 17 to 305, 18 to 60, 18 to 75, 18 to 250,18 to 400, 20 to 35, 20 to 60, 20 to 80, 20 to 175, 20 to 225, 20 to325, 20 to 400, 20 to 475, 25 to 45, 25 to 65, 25 to 100, 25 to 200, 25to 250, 25 to 300, 25 to 350, 25 to 400, 25 to 450, 30 to 280, 35 to250, 200 to 500, 200 to 400, 250 to 450, 250 to 350, or 300 to 400nucleotides in length).

In some embodiments, a spacer nucleic acid, sometimes referred to as aloop nucleic acid, can be positioned between the sense strand and theantisense strand. In some embodiments, the spacer nucleic acid can be anintron (see, for example, Wesley et al., 2001,The Plant J., 27:581-90).In some embodiments, although not required, the intron can be functional(i.e., in sense orientation; i.e., spliceable) (see, for example, Smithet al., 2000, Nature, 407:319-20). A spacer nucleic acid can be between20 nucleotides and 1000 nucleotides in length (e.g., 25-800, 25-600,25-400, 50-750, 50-500, 50-250, 100-700, 100-500, 100-300, 250-700,300-600, 400-700, 500-800, 600-850, or 700-1000 nucleotides in length).

In some embodiments, a construct can be produced by operably linking apromoter that is operable in plant cells; a DNA region, that, whentranscribed, produces an RNA molecule capable of forming a hairpinstructure; and a DNA region involved in transcription termination andpolyadenylation. It would be appreciated that the hairpin structure hastwo annealing RNA sequences, where one of the annealing RNA sequences ofthe hairpin RNA structure includes a sense sequence identical to atleast 20 consecutive nucleotides of the PR50 nucleotide sequence, andwhere the second of the annealing RNA sequences includes an antisensesequence that is identical to at least 20 consecutive nucleotides of thecomplement of the PR50 nucleotide sequence. In addition, as indicatedherein, the DNA region can include an intron (e.g., a functionalintron). When present, the intron generally is located between the twoannealing RNA sequences in sense orientation such that it is spliced outby the cellular machinery (e.g., the splicesome). Such a construct canbe introduced into one or more plant cells to reduce the phenotypicexpression of a PR50 nucleic acid (e.g., a nucleic acid sequence that isnormally expressed in a plant cell).

In some embodiments, a construct (e.g., an expression construct) caninclude an inverted-duplication of a segment of a PR50 gene, where theinverted-duplication of the PR50 gene segment includes a nucleotidesequence substantially identical to at least a portion of the PR50 geneand the complement of the portion of the PR50 gene. It would beappreciated that a single promoter can be used to drive expression ofthe inverted-duplication of the PR50 gene segment, and that theinverted-duplication typically contains at least one copy of the portionof the PR50 gene in the sense orientation. Such a construct can beintroduced into one or more plant cells to delay, inhibit or otherwisereduce the expression of a PR50 gene in the plant cells.

The components of a representative RNAi nucleic acid molecule directedtoward PR50 are shown in SEQ ID NO:4 (a sense strand to PR50); SEQ IDNO:5 (an antisense strand to PR50); and SEQ ID NO:6 (a spacer or loopsequence).

It would be appreciated by the skilled artisan that the region ofcomplementarity, between the antisense strand of the RNAi and the mRNAor between the antisense strand of the RNAi and the sense strand of theRNAi, can be over the entire length of the RNAi nucleic acid molecule,or the region of complementarity can be less than the entire length ofthe RNAi nucleic acid molecule. For example, a region of complementaritycan refer to, for example, at least 15 nucleotides in length up to, forexample, 500 nucleotides in length (e.g., at least 15, 16, 17, 18, 19,20, 25, 28, 30, 35, 49, 50, 60, 75, 80, 100, 150, 180, 200, 250, 300,320, 385, 420, 435 nucleotides in length up to, e.g., 30, 35, 36, 40,45, 49, 50, 60, 65, 75, 80, 85, 90, 100, 175, 200, 225, 250, 280, 300,325, 350, 400, 450, or 475 nucleotides in length). In some embodiments,a region of complementarity can refer to, for example, at least 15contiguous nucleotides in length up to, for example, 500 contiguousnucleotides in length (e.g., at least 15, 16, 17, 18, 19, 20, 25, 28,30, 35, 49, 50, 60, 75, 80, 100, 150, 180, 200, 250, 300, 320, 385, 420,435 nucleotides in length up to, e.g., 30, 35, 36, 40, 45, 49, 50, 60,65, 75, 80, 85, 90, 100, 175, 200, 225, 250, 280, 300, 325, 350, 400,450, or 475 contiguous nucleotides in length).

It would be appreciated by the skilled artisan that complementary canrefer to, for example, 100% sequence identity between the two nucleicacids. In addition, however, it also would be appreciated by the skilledartisan that complementary can refer to, for example, slightly less than100% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%sequence identity). In calculating percent sequence identity, twonucleic acids are aligned and the number of identical matches ofnucleotides (or amino acid residues) between the two nucleic acids (orpolypeptides) is determined. The number of identical matches is dividedby the length of the aligned region (i.e., the number of alignednucleotides (or amino acid residues)) and multiplied by 100 to arrive ata percent sequence identity value. It will be appreciated that thelength of the aligned region can be a portion of one or both nucleicacids up to the full-length size of the shortest nucleic acid. It alsowill be appreciated that a single nucleic acid can align with more thanone other nucleic acid and hence, can have different percent sequenceidentity values over each aligned region.

The alignment of two or more nucleic acids to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which allows alignments of nucleic acid orpolypeptide sequences to be carried out across their entire length(global alignment). Chenna et al., 2003, Nucleic Acids Res.,31(13):3497-500. ClustalW calculates the best match between a query andone or more subject sequences (nucleic acid or polypeptide), and alignsthem so that identities, similarities and differences can be determined.Gaps of one or more residues can be inserted into a query sequence, asubject sequence, or both, to maximize sequence alignments. For fastpairwise alignment of nucleic acid sequences, the default parameters canbe used (i.e., word size: 2; window size: 4; scoring method: percentage;number of top diagonals: 4; and gap penalty: 5); for an alignment ofmultiple nucleic acid sequences, the following parameters can be used:gap opening penalty: 10.0; gap extension penalty: 5.0; and weighttransitions: yes. For fast pairwise alignment of polypeptide sequences,the following parameters can be used: word size: 1; window size: 5;scoring method: percentage; number of top diagonals: 5; and gap penalty:3. For multiple alignment of polypeptide sequences, the followingparameters can be used: weight matrix: blosum; gap opening penalty:10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilicresidues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; andresidue-specific gap penalties: on. ClustalW can be run, for example, atthe Baylor College of Medicine Search Launcher website or at theEuropean Bioinformatics Institute website on the World Wide Web.

The skilled artisan also would appreciate that complementary can bedependent upon, for example, the conditions under which two nucleicacids hybridize. Hybridization between nucleic acids is discussed indetail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook etal. disclose suitable Southern blot conditions for oligonucleotideprobes less than about 100 nucleotides (Sections 11.45-11.46). The Tmbetween a nucleic acid that is less than 100 nucleotides in length and asecond nucleic acid can be calculated using the formula provided inSection 11.46. Sambrook et al. additionally disclose Southern blotconditions for oligonucleotide probes greater than about 100 nucleotides(see Sections 9.47-9.54). The Tm between a nucleic acid greater than 100nucleotides in length and a second nucleic acid can be calculated usingthe formula provided in Sections 9.50-9.51 of Sambrook et al.

The conditions under which membranes containing nucleic acids areprehybridized and hybridized, as well as the conditions under whichmembranes containing nucleic acids are washed to remove excess andnon-specifically bound probe, can play a significant role in thestringency of the hybridization. Such hybridizations and washes can beperformed, where appropriate, under moderate or high stringencyconditions. For example, washing conditions can be made more stringentby decreasing the salt concentration in the wash solutions and/or byincreasing the temperature at which the washes are performed. Simply byway of example, high stringency conditions typically include a wash ofthe membranes in 0.2×SSC at 65° C.

In addition, interpreting the amount of hybridization can be affected,for example, by the specific activity of the labeled oligonucleotideprobe, by the number of probe-binding sites on the template nucleic acidto which the probe has hybridized, and by the amount of exposure of anautoradiograph or other detection medium. It will be readily appreciatedby those of ordinary skill in the art that although any number ofhybridization and washing conditions can be used to examinehybridization of a probe nucleic acid molecule to immobilized targetnucleic acids, it is more important to examine hybridization of a probeto target nucleic acids under identical hybridization, washing, andexposure conditions. Preferably, the target nucleic acids are on thesame membrane. A nucleic acid molecule is deemed to hybridize to anucleic acid, but not to another nucleic acid, if hybridization to anucleic acid is at least 5-fold (e.g., at least 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 50-fold, or 100-fold) greater thanhybridization to another nucleic acid. The amount of hybridization canbe quantified directly on a membrane or from an autoradiograph using,for example, a Phosphorlmager or a Densitometer (Molecular Dynamics,Sunnyvale, Calif.).

A construct (also known as a vector) containing a RNAi nucleic acidmolecule is provided. Constructs, including expression constructs, aredescribed herein and are known to those of skill in the art. Expressionelements (e.g., promoters) that can be used to drive expression of aRNAi nucleic acid molecule are known in the art and include, withoutlimitation, constitutive promoters such as, without limitation, thecassava mosaic virus (CsMVM) promoter, the cauliflower mosaic virus(CaMV) 35S promoter, the actin promoter, or theglyceraldehyde-3-phosphate dehydrogenase promoter, or tissue-specificpromoters such as, without limitation, root-specific promoters such asthe putrescine N-methyl transferase (PMT) promoter or the quinolinatephosphosibosyltransferase (QPT) promoter. It would be understood by askilled artisan that a sense strand and an antisense strand can bedelivered to and expressed in a target cell on separate constructs, orthe sense and antisense strands can be delivered to and expressed in atarget cell on a single construct (e.g., in one transcript). Asdiscussed herein, a RNAi nucleic acid molecule delivered and expressedon a single strand also can include a spacer nucleic acid (e.g., a loopnucleic acid) such that the RNAi forms a small hairpin (shRNA).

Transgenic Plants and Methods of Making Transgenic Plants

Transgenic N. tabacum plants are provided that contain a transgeneencoding at least one RNAi molecule, which, when transcribed, silencesPR50 expression. As used herein, silencing can refer to completeelimination or essentially complete elimination of the PR50 mRNA,resulting in 100% or essentially 100% reduction (e.g., greater than 95%reduction; e.g., greater than 96%, 97%, 98% or 99% reduction) in theamount of PR50 polypeptide; silencing also can refer to partialelimination of the PR50 mRNA (e.g., eliminating about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50% or more of the PR50 mRNA), resulting in areduction (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%or more, but not complete elimination) in the amount of the PR50polypeptide.

A RNAi nucleic acid molecule can be transcribed using a plant expressionvector. Methods of introducing a nucleic acid (e.g., a heterologousnucleic acid) into plant cells (e.g., N. tabacum cells) are known in theart and include, for example, particle bombardment,Agrobacterium-mediated transformation, microinjection, polyethyleneglycol-mediated transformation (e.g., of protoplasts, see, for example,Yoo et al. (2007, Nature Protocols, 2(7):1565-72)), liposome-mediatedDNA uptake, or electroporation.

Following transformation, the transgenic plant cells can be regeneratedinto transgenic tobacco plants. The regenerated transgenic plants can bescreened for the presence of the transgene (e.g., a RNAi nucleic acidmolecule) and/or one or more of the resulting phenotypes (e.g., reducedamount of PR50 mRNA or PR50 polypeptide, reduced activity of a PR50polypeptide, reduced amount of nicotine or another alkaloid, and/orreduced amount of one or more TSNAs (in cured tobacco)) using methodsdescribed herein, and plants exhibiting the desired phenotype can beselected.

Methods of detecting alkaloids (e.g., nicotine) or TSNAs, and methods ofdetermining the amount of one or more alkaloids or TSNAs are known inthe art. For example, high performance liquid chromatography (HPLC)-massspectroscopy (MS) (HPLC-MS) or high performance thin layerchromatography (HPTLC) can be used to detect the presence of one or morealkaloids and/or determine the amount of one or more alkaloids. Inaddition, any number of chromatography methods (e.g., gaschromatography/thermal energy analysis (GC/TEA), liquidchromatography/mass spectrometry (LC/MS), and ion chromatography (IC))can be used to detect the presence of one or more TSNAs and/or determinethe amount of one or more TSNAs.

As used herein, “reduced” or “reduction” refers to a decrease (e.g., astatistically significant decrease), in green leaf or cured leaf, of/inone or more of the following: a) the amount of PR50 mRNA; b) the amountof PR50 polypeptide; c) the activity of the PR50 polypeptide; d) theamount of nicotine or another alkaloid. In addition, “reduced” or“reduction” refers to a decrease (e.g., a statistically significantdecrease), in cured leaf, in the amount of one or more tobacco-specificnitrosamines (TSNAs; e.g., N′-nitrosonornicotine (NNN),4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK),N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanal

(NNAL)). As used herein, “reduced” or “reduction” refers to a decreasein any of the above by at least about 5% up to about 95% (e.g., about 5%to about 10%, about 5% to about 20%, about 5% to about 50%, about 5% toabout 75%, about 10% to about 25%, about 10% to about 50%, about 10% toabout 90%, about 20% to about 40%, about 20% to about 60%, about 20% toabout 80%, about 25% to about 75%, about 50% to about 75%, about 50% toabout 85%, about 50% to about 95%, and about 75% to about 95%) relativeto similarly-treated leaf (e.g., green or cured) from a tobacco plantlacking the transgene. As used herein, statistical significance refersto a p-value of less than 0.05, e.g., a p-value of less than 0.025 or ap-value of less than 0.01, using an appropriate measure of statisticalsignificance, e.g., a one-tailed two sample t-test.

Leaf from progeny plants also can be screened for the presence of thetransgene and/or the resulting phenotype, and plants exhibiting thedesired phenotype can be selected. As described herein, leaf from suchtransgenic plants exhibit a reduced amount of nicotine or anotheralkaloid, or, in cured tobacco, a reduced amount of one or more TSNAs(e.g., compared to leaf from a plant lacking or not transcribing theRNAi). As described herein, transcription of the transgene results inleaf that exhibits a reduced amount of nicotine or another alkaloid, or,in cured tobacco, a reduced amount of one or more TSNAs relative to leaffrom a plant not transcribing the transgene. Leaf from regeneratedtransgenic plants can be screened for the amount of PR50, the amount ofone or more other intermediates in the biosynthesis of nicotine, theamount of nicotine, or, in cured tobacco, the amount of one or moreTSNAs, and plants having leaf that exhibit a reduced amount of nicotineor another alkaloid, or, in cured tobacco, a reduced amount of TSNAs,compared to the amount in a leaf from a corresponding non-transgenicplant, can be selected.

Transgenic plants exhibiting the desired phenotype can be used, forexample, in a breeding program. Breeding is carried out using knownprocedures. Successful crosses yield F₁ plants that are fertile and thatcan be backcrossed with one of the parents if desired. In someembodiments, a plant population in the F₂ generation is screened for thepresence of a transgene and/or the resulting phenotype using standardmethods (e.g., amplification, hybridization and/or chemical analysis ofthe leaf). Selected plants are then crossed with one of the parents andthe first backcross (BC₁) generation plants are self-pollinated toproduce a BC₁F₂ population that is again screened. The process ofbackcrossing, self-pollination, and screening is repeated, for example,at least four times until the final screening produces a plant that isfertile and reasonably similar to the recurrent parent. This plant, ifdesired, is self-pollinated and the progeny are subsequently screenedagain to confirm that the plant contains the transgene and exhibitsvariant gene expression. Breeder's seed of the selected plant can beproduced using standard methods including, for example, field testingand/or chemical analyses of leaf (e.g., cured leaf).

The result of a plant breeding program using the transgenic tobaccoplants described herein are novel and useful varieties, lines, andhybrids. As used herein, the term “variety” refers to a population ofplants that share constant characteristics which separate them fromother plants of the same species. A variety is often, although notalways, sold commercially. While possessing one or more distinctivetraits, a variety is further characterized by a very small overallvariation between individual with that variety. A “pure line” varietymay be created by several generations of self-pollination and selection,or vegetative propagation from a single parent using tissue or cellculture techniques. A “line,” as distinguished from a variety, mostoften denotes a group of plants used non-commercially, for example, inplant research. A line typically displays little overall variationbetween individuals for one or more traits of interest, although theremay be some variation between individuals for other traits.

A variety can be essentially derived from another line or variety. Asdefined by the International Convention for the Protection of NewVarieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov. 10,1972, On Oct. 23, 1978, and on Mar. 19, 1991), a variety is “essentiallyderived” from an initial variety if: a) it is predominantly derived fromthe initial variety, or from a variety that is predominantly derivedfrom the initial variety, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of the initial variety; b) it is clearly distinguishablefrom the initial variety; and c) except for the differences which resultfrom the act of derivation, it conforms to the initial variety in theexpression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety. Essentiallyderived varieties can be obtained, for example, by the selection of anatural or induced mutant, a somaclonal variant, a variant individualplant from the initial variety, backcrossing, or transformation.

Hybrid tobacco varieties can be produced by preventing self-pollinationof female parent plants (i.e., seed parents) of a first variety,permitting pollen from male parent plants of a second variety tofertilize the female parent plants, and allowing F₁ hybrid seeds to formon the female plants. Self-pollination of female plants can be preventedby emasculating the flowers at an early stage of flower development.Alternatively, pollen formation can be prevented on the female parentplants using a form of male sterility. For example, male sterility canbe produced by cytoplasmic male sterility (CMS), nuclear male sterility,genetic male sterility, molecular male sterility where a transgeneinhibits microsporogenesis and/or pollen formation, orself-incompatibility. Female parent plants having CMS are particularlyuseful. In embodiments in which the female parent plants are CMS, themale parent plants typically contain a fertility restorer gene to ensurethat the F₁ hybrids are fertile. In other embodiments in which thefemale parents are CMS, male parents can be used that do not contain afertility restorer. F₁ hybrids produced from such parents are malesterile. Male sterile hybrid seed can be interplanted with male fertileseed to provide pollen for seed set on the resulting male sterileplants.

Varieties and lines described herein can be used to form single-crosstobacco F₁ hybrids. In such embodiments, the plants of the parentvarieties can be grown as substantially homogeneous adjoiningpopulations to facilitate natural cross-pollination from the male parentplants to the female parent plants. The F₂ seed formed on the femaleparent plants is selectively harvested by conventional means. One alsocan grow the two parent plant varieties in bulk and harvest a blend ofF₁ hybrid seed formed on the female parent and seed formed upon the maleparent as the result of self-pollination. Alternatively, three-waycrosses can be carried out wherein a single-cross F₁ hybrid is used as afemale parent and is crossed with a different male parent. As anotheralternative, double-cross hybrids can be created wherein the F₁ progenyof two different single-crosses are themselves crossed.Self-incompatibility can be used to particular advantage to preventself-pollination of female parents when forming a double-cross hybrid.

The tobacco plants used in the methods described herein can be a Burleytype, a dark type, a flue-cured type, a Maryland type, or an Orientaltype. The tobacco plants used in the methods described herein typicallyare from N. tabacum, and can be from any number of N. tabacum varieties.A variety can be BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35,CC37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263,DF911, Galpao tobacco, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737,GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326,NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116,VA 119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171,KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944,msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297,NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH129, NC 2002, Neal Smith Madole, OXFORD 207, Perique tobacco, PVH03,PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81,RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179,Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, SpeightG-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR(Tom Rosson) Madole, VA 309, or VA359.

Nucleic acids that confer traits such as herbicide resistance (sometimesreferred to as herbicide tolerance), insect resistance, or stresstolerance, can also be present in the novel tobacco plants describedherein. Genes conferring resistance to a herbicide that inhibits thegrowing point or meristem, such as an imidazolinone or a sulfonylurea,can be suitable. Exemplary genes in this category encode mutant ALS andAHAS enzymes as described, for example, in U.S. Pat. Nos. 5,767,366 and5,928,937. U.S. Pat. Nos. 4,761,373 and 5,013,659 are directed to plantsresistant to various imidazolinone or sulfonamide herbicides. U.S. Pat.No. 4,975,374 relates to plant cells and plants containing a geneencoding a mutant glutamine synthetase (GS), which is resistant toinhibition by herbicides that are known to inhibit GS, e.g.phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,162,602discloses plants resistant to inhibition by cyclohexanedione andaryloxyphenoxypropanoic acid herbicides.

Genes for resistance to glyphosate also are suitable. See, for example,U.S. Pat. Nos. 4,940,835 and 4,769,061. Such genes can confer resistanceto glyphosate herbicidal compositions, including, without limitation,glyphosate salts such as the trimethylsulphonium salt, theisopropylamine salt, the sodium salt, the potassium salt and theammonium salt. See, e.g., U.S. Pat. Nos. 6,451,735 and 6,451,732. Genesfor resistance to phosphono compounds such as glufosinate ammonium orphosphinothricin, and pyridinoxy or phenoxy propionic acids andcyclohexones also are suitable. See, e.g., U.S. Pat. Nos. 5,879,903;5,276,268; and 5,561,236; and European Application No. 0 242 246.

Other suitable herbicides include those that inhibit photosynthesis,such as a triazine and a benzonitrile (nitrilase). See U.S. Pat. No.4,810,648. Other suitable herbicides include 2,2-dichloropropionic acid,sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylureaherbicides, triazolopyrimidine herbicides, s-triazine herbicides andbromoxynil. Also suitable are herbicides that confer resistance to aprotox enzyme. See, e.g., U.S. Pat. No. 6,084,155 and US 20010016956.

A number of genes are available that confer resistance to insects, forexample, insects in the order Lepidoptera. Exemplary genes include thosethat encode truncated Cry1A(b) and Cry1A(c) toxins. See, e.g., genesdescribed in U.S. Pat. Nos. 5,545,565; 6,166,302; and 5,164,180. Seealso, Vaeck et al., 1997, Nature, 328:33 -37 and Fischhoff et al., 1987,Nature Biotechnology, 5:807-813. Particularly useful are genes encodingtoxins that exhibit insecticidal activity against Manduca sexta (tobaccohornworm); Heliothis virescens Fabricius (tobacco budworm) and/or S.litura Fabricius (tobacco cutworm).

Mutant Plants and Methods of Making

Methods of making a N. tabacum plant having a mutation are known in theart. Mutations can be random mutations or targeted mutations. For randommutagenesis, cells (e.g., N. tabacum cells) typically are mutagenizedusing, for example, a chemical mutagen or ionizing radiation.Representative chemical mutagens include, without limitation, nitrousacid, sodium azide, acridine orange, ethidium bromide, and ethyl methanesulfonate (EMS), while representative ionizing radiation includes,without limitation, x-rays, gamma rays, fast neutron irradiation, and UVirradiation. The dosage of the mutagenic chemical or radiation isdetermined experimentally for each type of plant tissue such that amutation frequency is obtained that is below a threshold levelcharacterized by lethality or reproductive sterility. The number of M₁generation seed or the size of M₁ plant populations resulting from themutagenic treatments are estimated based on the expected frequency ofmutations. For targeted mutagenesis, representative technologies includeTALEN (see, for example, Li et al., 2011, Nucleic Acids Res., 39(14):6315-25) or zinc-finger (see, for example, Wright et al., 2005, ThePlant J., 44:693 -705). Whether random or targeted, a mutation can be apoint mutation, an insertion, a deletion, a substitution, orcombinations thereof, which are discussed in more detail below.

The resultant variety of Nicotiana tabacum includes plants having amutation in an endogenous PR50 nucleic acid (e.g., SEQ ID NO: 1, 2, or23) encoding a PR50 polypeptide sequence (e.g., SEQ ID NO: 3 or 24). Amutation in PR50 as described herein typically results in reducedexpression or activity of PR50, which, in turn, results in a reducedamount of nicotine or another alkaloid, or, in cured tobacco, a reducedamount of one or more TSNAs in the mutant plant relative to a plantlacking the mutation.

As discussed herein, one or more nucleotides can be mutated to alter theexpression and/or function of the encoded polypeptide, relative to theexpression and/or function of the corresponding wild type polypeptide.It will be appreciated, for example, that a mutation in one or more ofthe highly conserved regions would likely alter polypeptide function,while a mutation outside of those highly conserved regions would likelyhave little to no effect on polypeptide function. In addition, amutation in a single nucleotide can create a stop codon, which wouldresult in a truncated polypeptide and, depending on the extent oftruncation, loss of function.

Preferably, a mutation in a PR50 nucleic acid results in a tobacco plantthat exhibits reduced expression or activity of PR50, a reduced amountof nicotine or another alkaloid, or, in cured tobacco, a reduced amountof one or more TSNAs. Suitable types of mutations in a

PR50 coding sequence include, without limitation, insertions ofnucleotides, deletions of nucleotides, or transitions or transversionsin the wild-type PR50 coding sequence. Mutations in the coding sequencecan result in insertions of one or more amino acids, deletions of one ormore amino acids, and/or conservative or non-conservative amino acidsubstitutions in the encoded polypeptide. In some cases, the codingsequence of a PR50 comprises more than one mutation and/or more than onetype of mutation.

Insertion or deletion of amino acids in a coding sequence, for example,can disrupt the conformation of the encoded polypeptide. Amino acidinsertions or deletions also can disrupt sites important for recognitionof binding ligand(s) or substrate(s) or for activity of the polypeptide.It is known in the art that the insertion or deletion of a larger numberof contiguous amino acids is more likely to render the gene productnon-functional, compared to a smaller number of inserted or deletedamino acids. In addition, one or more mutations (e.g., a point mutation)can change the localization of the PR50 polypeptide, introduce a stopcodon to produce a truncated polypeptide, or disrupt an active site ordomain (e.g., a catalytic site or domain, a binding site or domain)within the polypeptide.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with a different amino acid residue having a similarside chain (see, for example, Dayhoff et al. (1978, in Atlas of ProteinSequence and Structure, 5(Suppl. 3):345-352), which provides frequencytables for amino acid substitutions), and a non-conservativesubstitution is one in which an amino acid residue is replaced with anamino acid residue that does not have a similar side chain.Non-conservative amino acid substitutions can replace an amino acid ofone class with an amino acid of a different class. Non-conservativesubstitutions can make a substantial change in the charge orhydrophobicity of the gene product. Non-conservative amino acidsubstitutions can also make a substantial change in the bulk of theresidue side chain, e.g., substituting an alanine residue for anisoleucine residue. Examples of non-conservative substitutions include abasic amino acid for a non-polar amino acid, or a polar amino acid foran acidic amino acid.

Simply by way of example, a PR50 amino acid sequence (e.g., SEQ ID NO:3)can be mutated to change tyrosine to histidine, which may change thesecondary structure of the polypeptide. In addition, a PR50 nucleic acidsequence (e.g., SEQ ID NO: 2) can be mutated to change the GG atposition 299 and 300 to GA or AG; or the C at position 91, 133, 178,208, or 409 to T, each of which would result in a stop codon. Such amutation would significantly reduce or essentially eliminate the amountof PR50 mRNA or polypeptide or the activity of PR50 in the plant.

Following mutagenesis, M₀ plants are regenerated from the mutagenizedcells and those plants, or a subsequent generation of that population(e.g., M₁, M₂, M₃, etc.), can be screened for those carrying a mutationin a PR50 sequence. Screening for plants carrying a mutation in a PR50nucleic acid or polypeptide can be performed directly using methodsroutine in the art (e.g., hybridization, amplification, nucleic acidsequencing, peptide sequencing, combinations thereof) or by evaluatingthe phenotype (e.g., reduced expression or activity of PR50, reducedamounts of nicotine or another alkaloid, and/or reduced amounts of oneor more TSNAs (in cured tobacco)). It would be understood that thephenotype of a mutant plant (e.g., reduced expression or activity ofPR50, reduced amounts of nicotine or another alkaloid, and/or reducedamounts of one or more TSNAs (in cured tobacco)) would be compared to acorresponding plant (e.g., having the same varietal background) thatlacks the mutation.

An M₁ tobacco plant may be heterozygous for a mutant allele and exhibita wild type phenotype. In such cases, at least a portion of the firstgeneration of self-pollinated progeny of such a plant exhibits a wildtype phenotype. Alternatively, an M₁ tobacco plant may have a mutantallele and exhibit a mutant phenotype (e.g., reduced expression oractivity of PR50, reduced amounts of nicotine or another alkalkoid,and/or reduced amounts of one or more TSNAs (in cured tobacco)). Suchplants may be heterozygous and exhibit a mutant phenotype due to aphenomenon such as dominant negative suppression, despite the presenceof the wild type allele, or such plants may be homozygous due toindependently induced mutations in both alleles.

As used herein, “reduced” or “reduction” refers to a decrease (e.g., astatistically significant decrease) in the expression or activity ofPR50, or in the amount of nicotine or another alkaloid, in either greenor cured tobacco, or in the amount of one or more TSNAs, in curedtobacco, by at least about 5% up to about 95% (e.g., about 5% to about10%, about 5% to about 20%, about 5% to about 50%, about 5% to about75%, about 10% to about 25%, about 10% to about 50%, about 10% to about90%, about 20% to about 40%, about 20% to about 60%, about 20% to about80%, about 25% to about 75%, about 50% to about 75%, about 50% to about85%, about 50% to about 95%, and about 75% to about 95%) relative tosimilarly-treated leaf (e.g., green or cured) from a tobacco plantlacking the mutation. As used herein, statistical significance refers toa p-value of less than 0.05, e.g., a p-value of less than 0.025 or ap-value of less than 0.01, using an appropriate measure of statisticalsignificance, e.g., a one-tailed two sample t-test.

A tobacco plant carrying a mutant allele can be used in a plant breedingprogram to create novel and useful lines, varieties and hybrids. Desiredplants that possess the mutation can be backcrossed or self-pollinatedto create a second population to be screened. Backcrossing or otherbreeding procedures can be repeated until the desired phenotype of therecurrent parent is recovered. DNA fingerprinting, SNP or similartechnologies may be used in a marker-assisted selection (MAS) breedingprogram to transfer or breed mutant alleles into other tobaccos, asdescribed herein.

In some embodiments, an M₁, M₂, M₃, or later generation tobacco plantcontaining at least one mutation is crossed with a second Nicotianatabacum plant, and progeny of the cross are identified in which themutation(s) is present. It will be appreciated that the second Nicotianatabacum plant can be one of the species and varieties described herein.It will also be appreciated that the second Nicotiana tabacum plant cancontain the same mutation as the plant to which it is crossed, adifferent mutation, or be wild type at the locus. Additionally oralternatively, a second tobacco line can exhibit a phenotypic trait suchas, for example, disease resistance, high yield, high grade index,curability, curing quality, mechanical harvesting, holding ability, leafquality, height, plant maturation (e.g., early maturing, early to mediummaturing, medium maturing, medium to late maturing, or late maturing),stalk size (e.g., small, medium, or large), and/or leaf number per plant(e.g., a small (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), orlarge (e.g., 16-21) number of leaves).

Cured Tobacco and Tobacco Products

The methods described herein allow for leaf constituents in a tobaccoplant to be altered while still maintaining high leaf quality. Asdescribed herein, altering leaf constituents refers to reducing, ingreen or cured leaf, the amount of nicotine or another alkaloid, orreducing, in cured leaf, the amount of one or more TSNAs. As describedherein, such methods can include the production of transgenic plants(using, e.g., RNAi or overexpression) or mutagenesis (e.g., random ortargeted).

Leaf quality can be determined, for example, using an Official StandardGrade published by the Agricultural Marketing Service of the USDepartment of Agriculture (7 U.S.C. §511); Legacy Tobacco DocumentLibrary (Bates Document #523267826/7833, Jul. 1, 1988, Memorandum on theProposed Burley Tobacco Grade Index); and Miller et al., 1990, TobaccoIntern., 192:55-7. For dark-fired tobacco, leaves typically are obtainedfrom stalk position C, and the average grade index determined based onFederal Grade and 2004 Price Support for Type 23 Western dark-firedtobacco.

Leaf from the tobacco described herein can be cured, aged, conditioned,and/or fermented. Methods of curing tobacco are well known and include,for example, air curing, fire curing, flue curing and sun curing. Agingalso is known and is typically carried out in a wooden drum (e.g., ahogshead) or cardboard cartons in compressed conditions for severalyears (e.g., 2 to 5 years), at a moisture content of from about 10% toabout 25% (see, for example, U.S. Pat. Nos. 4,516,590 and 5,372,149).Conditioning includes, for example, a heating, sweating orpasteurization step as described in US 2004/0118422 or US 2005/0178398,while fermenting typically is characterized by high initial moisturecontent, heat generation, and a 10 to 20% loss of dry weight. See, e.g.,U.S. Pat. Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. Thetobacco also can be further processed (e.g., cut, expanded, blended,milled or comminuted), if desired, and used in a tobacco product.

Tobacco products are known in the art and include any product made orderived from tobacco that is intended for human consumption, includingany component, part, or accessory of a tobacco product. Representativetobacco products include, without limitation, cigarettes, smokelesstobacco products, tobacco-derived nicotine products (e.g.,tobacco-derived nicotine pieces for use in the mouth), cigarillos,non-ventilated recess filter cigarettes, vented recess filtercigarettes, cigars, snuff, electronic cigarettes, electronic cigars,electronic cigarillos, e-vapor devices, pipe tobacco, cigar tobacco,cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, andcut tobacco. Representative smokeless tobacco products include, forexample, chewing tobacco, snus, pouches, films, tablets, sticks, rods,and the like. Representative cigarettes and other smoking articlesinclude, for example, smoking articles that include filter elements orrod elements, where the rod element of a smokeable material can includecured tobacco within a tobacco blend. In addition to thereduced-nicotine or reduced-TSNA tobacco described herein, tobaccoproducts also can include other ingredients such as, without limitation,binders, plasticizers, stabilizers, and/or flavorings. See, for example,US 2005/0244521, US 2006/0191548, US 2012/0024301, US 2012/0031414, andUS 2012/0031416 for examples of tobacco products.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1 Cloning of PR50

Using a subtractive hybridization screening strategy, a differentiallyexpressed PR50, 40S Ribosomal protein S12 homolog is identified fromBurley 21 root samples before and 3 days after topping (AF154659.1, Wanget al., 2000, Mol. Biol. Rep., 36:2285-9). A full length PR50 genomicfragment is cloned (SEQ ID NO:1) and sequence comparison shows it to bea single exon gene of 483 nucleotides (SEQ ID NO:2) coding for aputative protein product of 143 amino acids (SEQ ID NO:3).

SEQ ID NO: 1:TGACTAGCTCGTCGTATTGTGGGATGACGATACATCACCAGAATCAGTTAGCATGATACGCAGCTGGAATACCTCATCAAGATCAAAAGCTGGAGCTCCCCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGGCACGAGGCATTACTGCAACTCAAAGCAGATTGCGTCGTCTCTAAATTTTAAGGTTGCTGTGTTTTTTTGGGTTTAACATTTACCAAGAAAGAAATATGTCAGGAGAGGATGCTGCTGTTCCTGTTGTTGCTGCCGAGACTCCTGCTCCAGCACTTGGGGAGCCCATGGACATCATGACCGCTCTGCAGCTGGTGCTCAGGAAGTCTAAAGCTCATGGAGGACTTGCTCGAGGACTCCATGAAGGTGCTAAGGTGATTGAGAAGCATGCTGCGCAGCTTTGTGTGCTAGCAGAGGACTGCGACCAGCCAGACTATGTCAAGCTGGTCAAAGCTCTTTGTGCTGATCACAATGTCAGTTTGATTACAGTTCCCAATGCAAAAACTCTTGGCGAATGGGCTGGTTTATGCAAAATTGATTCTGAAGGGAAAGCAAGGAAAGTTGTTGGTTGTGGCTGTGTTGTCGTGAAGGATTATGGGGAAGAGACTGAGGGTCTCCATATCGTCCAGGAGTACGTGAAGTCTCATTAAATATAAGGTTGAGATGGAGCTTTAGGGGACTATGAGGCTAGATAAGTCTGAGACGGAGCTTTAGGGGGGAACTATGAAGCTAGAGATTCCATGAGACTATCTTTTTGGCATTTATTTAGAGTTGAATTTTTGAGATTTCAAACTATGTTCCCTTATTATTGTGTTACTTCAAGTTTTGTTTTACCTTCTGGGAAGATCTAATAGTTTGAAACTGCCGTCTAGTTTAAAAAAAAAAAAAAAAAAACTCGAGGGGGGGCCCGGTACCCAATTCTGAGTCGATTACATCCTGCGTCGTTACACGTCTGACTGAAACTGCGTACACTATCGCTGAACATCCCCTTTCGCACTGGGTAGCGAAAGCTC SEQ ID NO: 2ATGTCAGGAGAGGATGCTGCTGTTCCTGTTGTTGCTGCCGAGACTCCTGCTCCAGCACTTGGGGAGCCCATGGACATCATGACCGCTCTGCAGCTGGTGCTCAGGAAGTCTAAAGCTCATGGAGGACTTGCTCGAGGACTCCATGAAGGTGCTAAGGTGATTGAGAAGCATGCTGCGCAGCTTTGTGTGCTAGCAGAGGACTGCGACCAGCCAGACTATGTCAAGCTGGTCAAAGCTCTTTGTGCTGATCACAATGTCAGTTTGATTACAGTTCCCAATGCAAAAACTCTTGGCGAATGGGCTGGTTTATGCAAAATTGATTCTGAAGGGAAAGCAAGGAAAGTTGTTGGTTGTGGCTGTGTTGTCGTGAAGGATTATGGGGAAGAGACTGAGGGTCTCCATATCGTCCAGGAGTACGTGAAGTCTCATTAASEQ ID NO: 23ATGTCAGGAGAGGATGCTGCTGTTCCTGTTGTCGCTGCTGCCGAGACTCCTGCTCCAGCACTTGGGGAGCCCATGGACATCATGACCGCACTACAGCTGGTGCTAAAGAAGTCTAAAGCTCATGGAGGACTTGCTCGAGGACTCCATGAAGGTGCTAAGGTGATTGAGAAGCATGCTGCACAGCTTTGTGTGCTAGCTGAGGACTGTGACCAGCCAGATTACGTCAAACTGGTGAAAGCACTTTGTGCTGATCACAATGTCAGTTTAATTACAGTTCCCAATGCAAAAACTCTTGGCGAATGGGCTGGTTTATGCAAAATTGATTCTGAAGGGAAAGCAAGGAAGGTTGTTGGTTGTGGCTGTGTTGTCGTGAAGGATTATGGTGAAGAGACTGAGGGTCTCCATATCGTCCAAGAGTACGTGAAGTCTCATTA ASEQ ID NO: 3MSGEDAAVPVVAAETPAPALGEPMDIMTALQLVLRKSKAHGGLARGLHEGAKVIEKHAAQLCVLAEDCDQPDYVKLVKALCADHNVSLITVPNAKTLGEWAGLCKIDSEGKARKVVGCGCVVVKDYGEETEGLHIVQEYVKSH SEQ ID NO: 24MSGEDAAVPVVAAAETPAPALGEPMDIMTALQLVLKKSKAHGGLARGLHEGAKVIEKHAAQLCVLAEDCDQPDYVKLVKALCADHNVSLITVPNAKTLGEWAGLCKIDSEGKARKVVGCGCVVVKDYGEETEGLHIVQEYVKSH

Example 2 RNAi Construct

To study the function of PR50, a RNAi expression vector is constructedand transcribed in tobacco. The pBK-CMV cloning vector is used for theconstruction of an RNAi vector containing a 502 by sequence of PR50 inthe sense (SEQ ID NO:4) and antisense (SEQ ID NO:5) orientations. Thesetwo fragments are separated by a 660 bp Cax-2 spacer (SEQ ID NO:6).

SEQ ID NO: 4CGAGGCATTACTGCAACTCAAAGCAGATTGCGTCGTCTCTAAATTTTAAGGTTGCTGTGTTTTTTTGGGTTTAACATTTACCAAGAAAGAAATATGTCAGGAGAGGATGCTGCTGTTCCTGTTGTTGCTGCCGAGACTCCTGCTCCAGCACTTGGGGAGCCCATGGACATCATGACCGCTCTGCAGCTGGTGCTCAGGAAGTCTAAAGCTCATGGAGGACTTGCTCGAGGACTCCATGAAGGTGCTAAGGTGATTGAGAAGCATGCTGCGCAGCTTTGTGTGCTAGCAGAGGACTGCGACCAGCCAGACTATGTCAAGCTGGTCAAAGCTCTTTGTGCTGATCACAATGTCAGTTTGATTACAGTTCCCAATGCAAAAACTCTTGGCGAATGGGCTGGTTTATGCAAAATTGATTCTGAAGGGAAAGCAAGGAAAGTTGTTGGTTGTGGCTGTGTTGTCGTGAAGGATTATGGGGAAGAGACTGAGGGTCTCCATA TCGTCCSEQ ID NO: 5GGACGATATGGAGACCCTCAGTCTCTTCCCCATAATCCTTCACGACAACACAGCCACAACCAACAACTTTCCTTGCTTTCCCTTCAGAATCAATTTTGCATAAACCAGCCCATTCGCCAAGAGTTTTTGCATTGGGAACTGTAATCAAACTGACATTGTGATCAGCACAAAGAGCTTTGACCAGCTTGACATAGTCTGGCTGGTCGCAGTCCTCTGCTAGCACACAAAGCTGCGCAGCATGCTTCTCAATCACCTTAGCACCTTCATGGAGTCCTCGAGCAAGTCCTCCATGAGCTTTAGACTTCCTGAGCACCAGCTGCAGAGCGGTCATGATGTCCATGGGCTCCCCAAGTGCTGGAGCAGGAGTCTCGGCAGCAACAACAGGAACAGCAGCATCCTCTCCTGACATATTTCTTTCTTGGTAAATGTTAAACCCAAAAAAACACAGCAACCTTAAAATTTAGAGACGACGCAATCTGCTTTGAGTTGCAGTAAT GCCTCGSEQ ID NO: 6GAATTCGGTGAGTTCCCCCCTCCTCCCCTTTCACTTTTGTTTGTTGGTTTCTAAGTGCTCTTTCAATTTAGAGGTTGATGTTGGGAAATAATTAAACAATACTCTTGTTTTCTAAAATTTCTTGAAAACTACAATGTCTATAGAGGCAATATATTTGCTTCTAAACGTTGACGGTTTTGCAAGTCTTGCGGAGGAGCTTTGATCCAGTGTTAAAGAAATATATCATGTCTCTTATTCATCCTCCCTTTCTTTCCTTTGTGTTTTGCTTCACTCCTGGGGTTTCAACTTTTTTCTTTCCGTTTAACCTTTCCTTTTTTCTGCAGGATGGAACTTCAAATTACTTTAAAGGACTGATGCTCCTTCTCTGCTATTGATAGTTGCTGCAAGTTTCTTTGTGCATATAGATCCAGAGTCTATACGTAAGTTGTGTTTCTTTTTCGTGAAATTACCATATGACATTGACAGCTCCTGGTCTTCGTTTTATTTATTCTTTTGGTGTTCCTTTTAACCGATAACATCTGTTATTATTTCACTGTTACACTAATCTGCTTTGCTTATGGTCAGTCAGTTTAGCATTAGATTAGATAACCAGTTAACCATTTTGGGTCTCGTTAACGTAATATTGTATTGATAACTACCTTATCATATATATATCTCTGTTTTAGTGAATTC

The PR50 RNA vector is constructed as follows. The six hundred and sixtyby Cax2 sequence from BAC 57 intron 9 (SEQ ID NO:6) is cloned directlyinto pBK-CMK at the EcoRI site. XbaI and HindIII sites are added to the5′ and 3′ ends of a 502 bp sense-oriented PR50 sequence by means of PCRwith primers harboring these restriction enzymes sites (PMG526F: ATT CTAGAC GAG GCA TTA CTG CAA CTC A (SEQ ID NO:7) and PMG 526R: ATA AGC TTGGAC GAT ATG GAG ACC CTC A (SEQ ID NO:8)). Similarly, BamHI and SacIsites are created at the 5′ and 3′ ends of the corresponding PR50antisense fragment using PCR with primers harboring these restrictionenzyme sites (PMG 527F: ATG AGC TCC GAG GCA TTA CTG CAA CTC A (SEQ IDNO:9) and PMG 527R: ATG GAT TCG GAC GAT ATG GAG ACC CTC A (SEQ IDNO:10)) to produce pBK-CMV-PR50 RNAi plasmid.

To create a plant expression vector capable of mediating theconstitutive transcription of PR50 RNAi, the beta-glucuronidase ORF ofthe binary expression vector, pBI121 (Clontech) is excised and replacedwith the 502 bp XbaI-HindIII PR50 sense fragment, the 660 bp Cax2 spacercloned at the EcoRI site, and the 502 bp BamHI-SacI PR50 antisensefragment by cloning the RNAi nucleic acid molecule into the SbaI/SacIsties of PBI 121 to produce a plasmid, PBI121-PR50 RNAi.

Example 3 Transgenic Plants

TN90 cultivar is transformed and select first generation transformantsare propagated in the greenhouse. At the flowering stage, plants aretopped. Two weeks post-topping, the 3^(rd) and 4^(th) leaf from the topare collected, freeze dried and alkaloids are analyzed using GCMS.

Relative to controls, PR50 RNAi lines show significant reduction innicotine content (Table 1). Two years of field study of selectedtransgenic line and a control also show reduced nicotine content (FIG.3).

TABLE 1 First generation (T0) of transgenic plants showing reducednicotine Nicotine Nornicotine Anabasine Anatabine Total % nicotine PlantID (% by wt) (% by wt) (% by wt) (% by wt) alkaloids reduction GH22850.21 0.011 0.005 0.083 0.31 90.60 GH2289 0.26 0.015 0.010 0.208 0.4984.94 GH2261 0.41 0.021 0.007 0.174 0.61 81.12 GH2367 0.95 0.028 0.0040.030 1.01 69.01 GH2260 0.93 0.025 0.007 0.081 1.04 67.98 GH2368 1.200.031 0.006 0.046 1.28 60.55 GH2638 1.35 0.032 0.010 0.057 1.45 55.41GH2288 1.42 0.025 0.005 0.036 1.49 54.28 GH2280 1.54 0.029 0.005 0.0421.62 50.27 GH2639 1.43 0.041 0.017 0.211 1.70 47.72 TN90 2.49 0.0650.056 0.056 2.62 — Control 1 TN90 3.98 0.096 0.085 0.085 4.17 — Control2

Example 4 Quality of Leaf from Transgenic Plants

To compare leaf quality in existing low alkaloid tobacco lines with leafquality in PR50-silenced lines, plants from stable TN90 PR50 RNAi linesalong with K326, TN 90, B&W Low Nic, Burley 21 (Heggestad et al., 1960,University of Tennessee Agricultural Experiment Station, Bulletin 321;described therein as having reduced nicotine and nornicotine levels(nic1nic2 genotype)), HI Burley 21 or LI Burley 21 (Nielsen et al.,1988, Crop Science, 28:206; described therein as having intermediatelevels of total alkaloids), and LA Burley 21 (Legg et al., 1970, CropScience, 10:212; described therein as having “extremely low alkaloidcontent”) were grown in 1 plot rows with 3 replications. All plants weretopped at maturity, cured, and leaf samples were collected forevaluation. TN90 PR50 RNAi lines show significantly better leaf qualitycompared with the other low alkaloid lines. The data from the comparisonof nicotine levels in the TN90 PR50 RNAi lines with controls and anin-house low alkaloid line (TN90 PMT RNAi) with respect to leaf qualityis shown in FIG. 4.

Example 5 Random Mutagenesis

A novel genetic variation in a population of tobacco plants is createdto identify plants for low alkaloids. To induce random mutation,approximately 10,000 seeds of the selected tobacco variety are treatedwith 0.5% ethyl methane sulfonate (EMS; M1 seed), germinated andpropagated (into M1 plants). M2 seeds from self-pollinated M1 plants arecollected. A composite of M2 seed is grown and leaves from M2 plants arecollected and the DNA extracted. The PR50 sequence is amplified andsequenced, and analyzed for mutations.

Example 6 Targeted Mutagenesis Using TALENs

Transcription activator-like (TAL) effector protein sequences for PR50are designed (Table 2). The TALs are synthesized and cloned into a plantexpression vector (Life Technologies, Inc.) to serve as entry vectors.Depending on the intention, three different protocols are used togenerate mutagenic tobacco lines: a) one or more entry vectorscontaining the target TALs are directly transformed into tobaccoprotoplasts to generate random sequence deletion or insertion mutagenictobacco lines; b) a donor sequence (e.g., a reporter gene such as,without limitation, the GUS gene) flanked on the left and right sidewith sequences that are homologous to the target insertion sequence isco-transformed into tobacco protoplasts with one or more entry vectorsto generate mutagenic tobacco lines containing a target sequenceinterrupted by the donor sequence; or c) a donor sequence containingtarget TALs containing a point mutation is co-transformed into tobaccoprotoplasts with one or more entry vectors to generate tobacco lineshaving a point mutation within the target sequence.

TABLE 2 TALEN Sequences TALEN Target SEQ ID Name Gene LocationTarget Sequence NO: TALen- PR50 2-52 T GTCAGGAGAGGATGCT gctgttcctgttgtt19 PR1 GCTGCCGAGACTCCTGCTCC A TALen- PR50 33-101 T GCTGCCGAGACTCCTGCTCC20 PR2 agcacttggggagcccatggacatcatga  CCGCTCTGCAGCTGGTGCTC A TALen- PR502-55 T GTCAGGAGAGGATGC tgctgttcctgttgtcgct 21 PR3 homologueGCTGCCGAGACTCCTGCTCC A TALen- PR50 126-194  T GCTGCCGAGACTCCTGCTCC 22PR4 homologue agcacttggggagcccatggacatcatga CCGCTCTGCAGCTGGTGCTC A

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

What is claimed is:
 1. A variety of Nicotiana tabacum, the varietycomprising plants having a mutation in an endogenous nucleic acid, thewild type endogenous nucleic acid encoding the PR50 sequence shown inSEQ ID NO:2.
 2. The variety of claim 1, wherein material from the mutantplants, when cured, exhibits a reduced amount of at least one TSNArelative to cured material from a plant lacking the mutation.
 3. Thevariety of claim 1, wherein leaf from the mutant plants exhibits areduced amount of nicotine relative to leaf from a plant lacking themutation.
 4. The variety of claim 3, wherein leaf from the mutant N.tabacum plant exhibits comparable or better quality than leaf from theplant lacking a mutated PR50 sequence.
 5. Cured leaf from the N. tabacumvariety of claim
 1. 6. A tobacco product comprising the cured leaf ofclaim
 5. 7. The tobacco product of claim 6, selected from the groupconsisting of cigarettes, smokeless tobacco products, tobacco-derivednicotine products, cigarillos, non-ventilated recess filter cigarettes,vented recess filter cigarettes, cigars, electronic cigarettes,electronic cigars, electronic cigarillos, e-vapor devices, snuff, pipetobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaftobacco, shredded tobacco, and cut tobacco.
 8. A method of making aNicotiana tabacum plant, comprising: inducing mutagenesis in N. tabacumcells to produce mutagenized N. tabacum cells; obtaining one or more N.tabacum plants from the mutagenized N. tabacum cells; and identifying atleast one of the N. tabacum plants that comprises a mutated PR50sequence.
 9. The method of claim 8, further comprising identifying atleast one of the N. tabacum plants that exhibits reduced amounts ofnicotine relative to a N. tabacum plant lacking a mutated PR50.
 10. Themethod of claim 8, further comprising identifying at least one of the N.tabacum plants that, when material from the at least one plant is cured,exhibits a reduced amount of at least one TSNA relative to curedmaterial from a N. tabacum plant lacking a mutated PR50.
 11. The methodof claim 8, wherein leaf from the mutant N. tabacum plant exhibitscomparable or better quality than leaf from the plant lacking a mutatedPR50 sequence.
 12. The method of claim 8, wherein the N. tabacum plantis a Burley type, a dark type, a flue-cured type, or an Oriental type.